Project Summary Our inability to culture hematopoietic stem cells (HSCs), or study physiologic and pathologic hematopoiesis in vitro, remain as significant problems in hematopoietic biology. Recent studies focus on reprogramming pluripotent stem cells (PSCs) or somatic cells to hematopoietic stem and progenitor cells (HSPCs). These studies, however, rely on gene delivery systems that integrate into the host genome. This widens the gap between the bench and the bedside by impeding the application of these in vitro generated blood products for transplants and drug testing platforms. A method to generate HSPCs de novo without genomic disruption, and use of these studies for disease modeling, are critical for understanding the mechanisms behind the pathologic hematopoiesis encountered in multiple hematopoietic disorders such as Fanconi Anemia (FA). In this disorder, the associated bone marrow failure (BMF) is preceded by a significant reduction in CD34+ hematopoietic progenitors in utero. Using a minimal set of transcription factors (TFs) we have shown our ability to induce a hemogenic program in mouse fibroblasts that generate HSC-like cells over time that are phenotypically and functionally similar to HSCs. Promising results after translating these methods to human dermal fibroblasts (HDFs) can provide a novel model system of FA and other hematologic disorders in vitro for drug testing and gene editing platforms with the goal of therapeutic discovery and eventual patient-specific HSC transplants. The aims of this F31 application proposal are to 1) investigate non-integrative methods to induce a hemogenic program in human fibroblasts and 2) utilize hemogenic reprogramming to study the pathologic hematopoiesis that precedes BMF in the FA disease state. The ability to generate zero footprint hematopoietic cells de novo (and therefore without the risk of insertional mutagenesis and oncogenesis) is highly clinically relevant, as are any discoveries made regarding defective hematopoiesis in FA after application of this novel reprogramming strategy. To achieve these goals, I will first generate polycistronic cassettes carrying our TF cocktail for hemogenic induction that will then be transferred to a self-replicating RNA (srRNA) system that robustly expresses these factors without genomic disruption. HDFs will then be reprogrammed with these constructs to ensure efficient hemogenic induction. I will also induce hemogenesis in patient-specific FA HDFs to determine the impact of the FA pathway defect in emerging hematopoiesis. I will rigorously analyze global gene expression profiles (RNAseq) and TF binding for candidate pathways (ChIP-PCR) in our reprogrammed cells to identify potential mechanisms behind the FA pathway in definitive hematopoiesis. The strengths of this proposal lie in the innovative reprogramming strategy that recapitulates definitive hematopoiesis in a dish, and the potential findings after applying this technology to hematologic disease models. This project is designed to frame the research in a clinical context and provide specialized training of a future physician scientist.